The lipoproteins, which include chylomicrons, very-low-density lipoproteins (VLDL), low-density-lipoproteins (LDL), and high-density-lipoproteins (HDL), are the primary carriers of plasma cholesterol. These particles are composed of various proportions of triglycerides, cholesterol, cholesterol ester, phospholipids and proteins. The latter are known as apolipoproteins and play a key role in the metabolism of lipoproteins. Some activate enzymes that are important in the covalent modification of lipids and in the remodeling of lipoprotein subfractions while others serve as receptor ligands that target remodeled lipoproteins to specific tissue sites where their respective lipid components are stored or used.
Lipoprotein(a) is a class of lipoprotein particles similar to LDL, but distinct due to the covalent linkage of apo B100 to apolipoprotein(a) ("apo(a)"), a glycoprotein with significant homology to plasminogen. High plasma concentrations of Lp(a) are associated with an increased risk of atherosclerotic disorders including intermittent claudication, aortic aneurysms, coronary artery stenosis, myocardial infarction and cerebral infarction. Studies implicating the role of Lp(a) in atherogenesis have focused on the binding of Lp(a) to endothelial cells and macrophages and to extracellular plasma proteins such as fibrin. In vitro studies with human fibroblasts and monocytes have demonstrated that Lp(a) is taken up by the LDL receptor. In addition, lipid peroxidation of Lp(a) results in uptake by the scavenger receptor on macrophages. The scavenger receptor is structurally different than the LDL receptor and is thought to play a role in lipid peroxide modified LDL uptake in atheromas. It has been demonstrated by immunohistochemistry that apo(a) and apo B are present in arterial wall plaques. Lp(a)-like particles can be isolated from plaques. Furthermore, a correlation between serum Lp(a) levels and amounts of apo(a) in arterial walls has been reported.
Some studies have implicated a role for Lp(a) in atherothrombosis due to the homology between apo(a) and plasminogen. Lp(a) competes with plasminogen for binding to fibrin. Because Lp(a) does not enzymatically cleave fibrin, it could inhibit fibrin clot dissolution. Thrombus formation in the intra-coronary arteries is thought to be the major cause of myocardial infarction. Thus, Lp(a) may have a multimodal mechanism in atherogenesis.
Apo(a) is highly heterogeneous in size with reports of 19 to 34 different alleles (Lackner, C. et al., J. Clin. Invest., 87:2158-61 (1991); Kamboh, M. et al., Am. J. Hum. Genet., 49:1063-74 (1991); Marcovina, S. et al., Biochem. Biophys. Res. Comm., 191:1192-6 (1993)). Utermann and co-workers (J. Clin. Invest. 80:458-67 (1987) and Sandholzer et al., Arterio and Thromb. 12:1214-26 (1992)) have designated six different isoform categories, and Marcovina et al. (Arterio and Thromb. 13:1037-45 (1993)) have added a seventh according to electrophoretic mobility compared with that of apo B. The assigned approximate molecular weights to each category are listed in Table 1 below. Apo(a) polymorphism is due to a series of alleles each coding for isoforms differing in the number of Kringle 4 domains (structurally similar to Kringle 4 in plasminogen). Apo(a) contains 5 to 37 Kringle 4 repeats, one Kringle 5 domain and an inactive serine protease region which has 94% homology to plasminogen, as seen in FIG. 1. Thus, size differences in the apo(a) phenotypes are due primarily to the number of Kringle 4 repeat units in apo(a), although differences in glycosylation may also contribute.
TABLE 1 ______________________________________ Approximate Molecular Weights of Apo(a) Isoform Categories per Utermann et al. Isoform Category Approximate apo(a) MW ______________________________________ F 400,000 B 460,000 S1 520,000 S2 580,000 S3 640,000 S4 700,000 S5 760,000 ______________________________________
Genetic size polymorphisms are associated with plasma Lp(a) concentrations. Low molecular weight isoform categories (F, B, S1 and S2) are associated with high Lp(a) concentrations and high -molecular weight isoform categories (S3, S4 and S5) are associated with low plasma Lp(a) concentrations (Gaubatz, J. et al., J. Lipid Res. 31:603-13 (1990)). Thus, Lp(a) concentrations are thought to be genetically regulated. Furthermore, because elevates Lp(a) levels are associated with increased risk of atherosclerotic diseases, an association has been reported between apo(a) isoform category and risk of coronary artery disease.
Several methods have been developed over the past several decades to measure Lp(a). Initially, Lp(a) was identified by electrophoresis in starch or agar gels under nondenaturing conditions and lipid-binding stains were used for visualization. However, this method was qualitative, not quantitative. Radial immunodiffusion (RID), electroimmunodiffusion (EID), and immunoelectrophoresis (IEP) methods were developed when purified antibodies became available. RID lacked the sensitivity required to measure Lp(a) in all serum and plasma samples and, more importantly, was influenced by the differences in the Lp(a) particle size. However, both EID and IEP, used in the majority of studies associating increased Lp(a) with risk of cardiovascular disease, are accurate and sensitive. Nevertheless, these methods are laborious, time-consuming, and not well suited for studies involving a large number of samples. In addition, neither method lends itself to automation. Immunoturbidimetric and immunonephelometric methods are affected by high concentrations of triglycerides and by freezing the sample. Additionally, the nephelometric method is also highly sensitive to differences in the size of the Lp(a) particle being measured because of the accompanying differences in light-scattering properties. Radioimmunoassays (RIAs) are both sensitive and specific; however, the radioactive component has a limited shelf-life and requires dedicated equipment as well as special handling.
To overcome the problems associated with these methods, the immunoassay known as the sandwich ELISA was developed. However, the ELISA method must be applied with an understanding of the unique molecular characteristics of the Lp(a) particle, which is heterogeneous in size and density. One type of commercially available Lp(a) ELISA assay makes use of a mouse monoclonal anti-apo(a) antibody as the capture antibody and a sheep polyclonal anti-apo B-peroxidase conjugate as the detection system.
Any immunoassay method used to quantify Lp(a) should employ an antibody that recognizes all isoforms equally well; thus, only an antibody that recognizes a non-repetitive epitope within the apo(a) molecule and that does not occur within the plasminogen molecule should be employed in the assay. Since the Kringle 4 domains of apo(a) are highly repetitive, the epitope should exist within either the Kringle 5 or protease-like domains. However, according to J. E. Tomlinson et al. (J. Biol. Chem. 264:5957-65,1989)), rhesus monkey apo(a) does not contain a Kringle 5 domain; therefore, in order to develop an assay for the quantitation of Lp(a) in this common animal model and, possibly, other Old World monkey species (baboons, African green and cynomologous monkeys), the antibody employed must recognize a unique, non-repetitive epitope within only the protease domain of apo(a), i.e., one that does not occur in the plasminogen molecule. Because the protease domain of human apo(a) has a 94% homology to human plasminogen, the likelihood of obtaining an antibody which recognizes a non-repetitive epitope unique to only apo(a) is quite small.
Such an antibody has been developed and is in use in the commercially available ELISA Lp(a) assay mentioned above. However, it would be desirable to know the exact amino acid sequence of the reactive epitopes so that assays using such epitopes or peptides to detect Lp(a) can be developed. The DNA nucleotides that code for these peptides, when reproduced, could be used in nucleic acid based detection and amplification technologies to detect or quantitate apo(a). Also, antibodies against apo(a), both monoclonal and polyclonal, could be raised when such a peptide is used as an antigen in a suitable animal.